Hemocyanins are the principal serum proteins of many mollusks and arthropods. Hemocyanin is a blue copper protein responsible for the transport of oxygen. Its concentration in the blood is 20-120 mg/ml. It occurs among mollusks in snails (class of Gastropoda), inkfishes (class of Cephalopoda), chitons (class of Polyplacophora), elephant tooth snails (class of Scaphopoda) and mussels (class of Bivalvia). It has been found among arthropods in scorpions and spiders (class of Arachnida), horseshoe crabs (class of Xiphosura), shrimps, lobsters, crabs and crayfishes (class of Crustacea) and centipedes (class of Myriopoda).
Arylphorin is a principal protein of insects (class of Hexapoda) and is characterized by its high phenylalanine and tyrosine content. A concentration of up to 60 mg/ml is found in the larvae and pupae of holometabolic insects and in lower concentrations also in adult Holometabola and Hemimetabola. There is a special name for the arylphorins of each animal species (drosophilin, calliphorin, etc.). Arylphorins do not bind oxygen. Various functions as transporters of substances and cuticular components have been discussed. It was recently found, for example, in the course of studies on monoclonal antibodies, that arylphorins are structurally homologous with the hemocyanins of arthropods and are probably derivatives thereof.
Hemocyanins and arylphorins are synthesized in special cells. These cells, at least during their biogenesis, do not float freely in the blood circulation, but are associated with certain tissues that are in contact with the blood (cardiac muscle septa, intestinal gland, fat body, sinus of the eye, etc.). Hemocyanins and arylphorins are concentrated in these cells and released into the blood. Native snail hemocyanin is cylindrical, and with 35 nm has the volume of a polio virus. The other mollusk hemocyanins are half this size. Arthropod hemocyanins and arylphorins are structurally based on a cube with a side length of 10 nm. This cube may be halved in arylphorins, while in hemocyanins it is often oligomerized. The largest arthropod hemocyanin (Limulus) consists of 8 cubes and has a side length of 25 nm, which roughly corresponds to the size of a ribosome.
Arthropod hemocyanins and arylphorins consist of up to 8 immunologically different subunits having a molecular weight of around 70-80,000. The 10 nm basic cube is a hexamer made up of such subunits. Each type of subunit plays a specific structural part in oligohexameric hemocyanins. There are several complete sequence analyses in existence, as well as a detailed roentgen structural model. Many details of the genetic structure of arylphorins are known.
Hemocyanins and arylphorins can be broken down into subunits by dialysis against an alkaline pH and removal of divalent cations. In mollusks, this subunit consists of 7-8 globular domains, each of which has a molecular weight of around 55,000. The domains are immunologically very different. One of them has been completely sequenced. Each has an active, oxygen-binding center, consisting of 2 copper atoms. Up to 160 such domains are present in the native molecule. Their arrangement in the 35 nm particle is known in detail.
Based on the structural heterogeneity described, and its completely xenogeneic nature, since mollusks and arthropods have been separated from vertebrates as protostomia for at least 600 million years, hemocyanin is one of the strongest antigens known. In mammalians it leads to the formation of a very powerful antiserum, moves the T4/T8 ratio in favor of the T4 helper cells and at the site of application leads to local erythema and invasion by macrophages. This has been used for many years in immunologic research with keyhole limpet hemocyanin (KLH).
It is known that KLH gives rise to cytotoxic T-cells and has distinct antitumor effects in vitro and in various animal models in vivo. This quality of KLH has been repeatedly demonstrated. Clinical treatment with KLH leads to a significant drop in the rate of recurrence in cases of superficial bladder carcinoma, while unpleasant side effects have never been observed, and other types of tumors have also been positively affected (C. D. Jurincic et al. Uroscope, Information u. Fortbildung i.d. Urologie, issue 1, 1986). The exact mechanism of action of KLH on bladder carcinoma cells is not as yet entirely clear. It has been found, however, that the site of action is completely different from existing chemotherapeutic sites. The results of chemotherapy are partially contradictory. While cytotoxic drugs attack all urothelial cells, KLH stimulates the macrophages of the entire organism (J. E. Curtis et al. "Antigen dose in human immune response relationships in the human immune response to keyhole-limpet hemocyanin." Journal of Laboratory in Clin. Med., 78:61 1971). Curtis has shown that KLH brings about both a primary cell-mediated, delayed hypersensitivity and a primary antibody response in humans.
This potent antigen thus acts as a nonspecific stimulant that activates T-lymphocytes and lymphokine-producing macrophages.
It was the task of this invention to make available additional powerful antigens of the same class of substances with an equal or stronger effect.
The task is solved by use of hemocyanin, other than keyhole-limpet hemocyanin, and/or arylphorin, to influence the immune system and for the treatment of tumors.